The dopamine transporter (DAT) mediates the inactivation of released dopamine through its reuptake. DAT is the major molecular target responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. Homologous neurotransmitter transporters for serotonin and norepinephrine are targets for antidepressant medications as well as secondary targets for psychostimulants. The long-term goals of this research are to determine the structural bases of substrate translocation and drug binding to DAT and eventually to understand whether the action of cocaine can be prevented while simultaneously preserving dopamine transport. During this grant period we showed that: substrates and non-substrate inhibitors differentially alter the conformations of DAT; within the class of inhibitors, drug-specific conformational changes are detectable and may be consistent with the drugs' distinct behavioral effects; DAT is an oligomer with a symmetrical interface in TM6 and a separate symmetrical interface in TM4, and that cocaine binding alters the TM4 interface and possibly the quaternary structure of DAT; amphetamine and cocaine redistribute DAT to/from the cell surface in opposite ways; N-terminal phosphorylation of DAT occurs in response to PKC activation, but this phosphorylation is not necessary for PKC-induced DAT internalization. We now propose to test the following hypotheses: a) Particular transmembrane segments line the transport pathway of DAT. b) The cocaine binding site is lined by residues from the third intracellular loop (IL3) as well as the transmembrane segments, c) The orientations of cocaine analogs and benztropine analogs bound within the transporter are different, and they induce/stabilize different conformations of DAT. d) Cytoplasmic loops of the transporter form part of a gate that alternately blocks and permits access from the transport pathway to the intracellular milieu. To test these hypotheses, we propose studies with the following specific aims: 1) To determine the residues in TM1, TM3, and TM5 of DAT that are water-accessible and likely to line the transport pathway. 2) To identify residues in and very near to the binding site of a cocaine analog using sulfhydryl-targeted affinity labeling. 3) To compare sulfhydryl-targeted affinity labeling using cocaine and benztropine analogs in which the sulfhydryl reactive groups have been placed in different positions on the tropane rings. 4) To map the intracellular and extracellular accessibility during different functional states of substituted-cysteines through IL1 and IL3 of DAT.